Background: Myostatin (Mstn), a member of the TGF-β superfamily, is a negative regulator of skeletal muscle mass in mammals. Precise regulation of Mstn expression is important for somite growth in fish. MicroRNA (miRNA), a type of small non-coding RNA, regulates gene expression at the post-transcriptional level and participates in various physiological functions. A growing amount of evidence has emphasized the importance of miRNA in the development of skeletal muscle.Aims: This study aims to study how miRNAs regulate myostatin b (mstnb) post-transcriptionally in tilapia.Methods/Results:Mstnb 3′ UTR sequences were obtained, and the results of tissue distribution showed that mstnb was expressed in several tissues, including brain, white muscle, gut, and adipose tissue. A total of 1,992 miRNAs were predicted to target mstnb in tilapia using bioinformatics, and a dual-luciferase reporter experiment confirmed that miR-181a/b-5p, miR-30-3p, miR-200a, and miR-27a were the target miRNAs of mstnb. Mutagenesis of the miR-181b-5p binding sites of mstnb significantly increased the luciferase signal compared to the wild-type mstnb. In tilapia primary muscle cells, overexpression of miR-181b-5p led to the downregulation of MSTNb expression, and the inhibitory effect of MSTNb on the downstream genes was dismissed, while inhibition of miR-181b-5p could reverse these phenomena.Conclusion: Taken together, our results suggested that miR-181b-5p could promote the growth of skeletal muscle by decreasing the MSTNb protein level in tilapia. Genetic Improvement of Farmed Tilapia (GIFT), a freshwater fish with a fast growth rate and high disease resistance, is a popular aquaculture fish worldwide that provides premium protein for people. Since the major edible part of the fish is skeletal muscle, the fish growth performance is mainly determined by the development of skeletal muscle. Skeletal muscle development is an accurate process that is regulated by positive factors, including muscle-specific myogenic regulatory transcription factors (MRFs)  and negamstn is specifically expressed in skeletal muscle, while mstn is widely distributed in teleosts. In addition, mstn has several different types in teleosts as a result of gene duplication. According to the genome listed in the NCBI, for example, mstn-1 (or mstnb) and mstn-2 (or mstna) exist in Nile tilapia. Tissue distribution revealed that mstnb is mainly expressed in the brain, eye, gill, gut, and skeletal muscle in Nile tilapia  superfamily, is regarded as a specific muscle negative regulatory factor . Mstn, a tilapia . Mstn caNA level , 6. MoreNA level . Regardlmstn promoter  transcription factors , were found in the promoter , 9. The promoter , and thepromoter . On the promoter . MiRNAs promoter , 15. Sevpromoter . For exae 3′ UTR . There ie 3′ UTR . In mamme 3′ UTR –22. For e 3′ UTR . In tele chuatsi . MiRNAs mstn were predicted based on the miRNA transcriptome database. We screened the miRNAs that targeted mstn using the dual-luciferase reporter system and verified the regulation of miRNA on mstn in tilapia primary muscle cells. The objective of this study was to find miRNAs that target mstn and regulate the growth of tilapia. Clarifying the regulatory mechanism of mstn using miRNA for skeletal muscle growth would help deepen the understanding of tilapia growth. In addition, it is a new paradigm to study miRNA in fish with economic value. This could increase economic benefits and make an important contribution to the aquaculture industry.In our previous study, a deep sequencing of the Nile tilapia miRNA transcriptome was conducted in our lab . In thisTilapia were obtained from the local farm of Guangdong Tilapia Breeding Farm. They were maintained in a water circulation system with water temperature at 28°C under a 12/12 h light/dark photoperiod. The fish were fed to satiety daily with commercial extruded feed . The time of domestication was longer than 1 week. They were narcotized with eugenol before decollating. Skeletal muscle samples were collected from fish weighting 6–8 g.mstnb and mstna 3′ UTR were obtained using PCR with KOD neo plus . To predict miRNAs that potentially bind to mstnb, a tilapia miRNA transcriptome was conducted (data not shown) and the PITA targets (http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html) were queried . These reporters and miRNA mimics  were co-transfected into HEK 293T cells, and the relative luciferase activity was detected using the Luciferase Assay Systems kit  according to the manufacturer's protocol. All primers are listed in A recombined psiCHECK2 vector  containing the mstnb/a and miR-181b-5p was assayed using real-time PCR.For miR-181b-5p cloning, the specific primers were designed according to the miRNA transcriptome, and the sequence of miR-181b-5p was further verified. For tissue distribution, total RNA was extracted from the tissue samples of the telencephalon, diencephalon, cerebellum, medulla oblongata, spinal cord, hypothalamus, pituitary, gill, heart, liver, spleen, stomach, foregut, midgut, hindgut, adipose tissue, red muscle, white muscle, testis, and kidney of three adult male tilapia (BW 150–180 g). All samples were snap-frozen in liquid nitrogen once removed, followed by storage at −80°C until RNA extraction. After RNA extraction and reverse transcription, the tissue distribution of n = 3–4) was extracted from primary muscle cells using Trizol reagent , and the RNA concentration was determined using a Nanodrop200C spectrophotometer . A total of 1 μg of total RNA was reverse-transcribed into cDNA with the M-MLV Reverse Transcriptase , and the PCR reaction was amplified with the Thunderbird SYBR Green qPCR Mix  according to the manufacturer's protocol. For mRNA quantitative analysis, β-actin was detected as the internal normalization control. Specifically, miRNA stem-loop primers were used in reverse transcription and U6 snoRNA was used as the internal control. Gene expression was normalized against the expression of the control using the comparative Ct method  and protease inhibitor  were added to a cell lysis reagent radio immunoprecipitation assay buffer  at a ratio of 1:100. First, the concentration of protein was determined, and 10 μg total protein was separated on an SDS-PAGE and transferred to a polyvinylidene fluoride membrane . Next, 5% BSA (dissolved in TBST) was used to block the membrane for an hour at room temperature, and the specific antibody was incubated at 4°C overnight. The internal control was β-actin  or GAPDH . Third, HRP-conjugated goat anti-rabbit or anti-mouse IgG antibody  was incubated for an hour at room temperature after washing the membrane three times (10 min each). Finally, the membranes were washed, and the immunoreactivity was determined by an enhanced chemiluminescence ECL detection kit . The gray intensity analysis was conducted using Image J 1.45 .n = 25–30) and collected in an ice-cold isolation medium . After removing the remaining red muscle and skin, the samples were sliced and hydrolyzed with collagenase  at 28°C for 20 min. After washing twice with washing medium (isolation medium without horse serum), trypsin  was used to digest the remaining sample at 28°C for 20 min. This mixture was diluted with additional isolation medium (1:4) to neutralize the digestion of trypsin. After centrifugation, the cells were resuspended with complete medium (washing medium with 10% FBS) and filtered through a sterile nylon sieve . Then, the myosatellite cells were collected in complete medium, and seeded onto 24-well or 12-well plates  at a density of 4 × 105 cells/cm2 for different experiments. After 24 h, the adhered myosatellite cells were covered with fresh complete medium. To further verify the adhered myosatellite cells, an immunofluorescence experiment with MyoD  and MyoG  antibody was conducted as previously described  into primary muscle cells using Lipofectamine3000  at 80 nM for 24 h, and the inverted fluorescent microscope ECLIPSE Ti-E  was used to observe the fluorescence. Transfection efficiency was defined as the ratio of cells in red (CY3-labeled) and number of cells in blue (DAPI stained). MiRNA mimics and antagomir  were used in the overexpression and inhibition experiments. The negative control was a scrambled RNA duplex that was not homologous to the tilapia genome. All oligonucleotides were 2′-OMe modified, and the end of the antagomir was conjugated to cholesterol. MiRNA mimics were transfected into the dispersed cells at 80 nM for 24 h, while antagomir was incubated at 100 nM for 24 h, and cells were harvested for RNA extraction or western blot. Each experiment was repeated three times independently.p < 0.05. p < 0.05 was noted with *p < 0.01 with **, and p < 0.001 with ***.Data are expressed as the means ± SEM unless otherwise stated. Statistical significance was assessed by one-way ANOVA followed by Bonferroni's multiple comparison tests. Statistical significance was defined as Mstna and Mstnb were cloned. The results showed that the Mstnb 3′ UTR was 1,307 bp, which was consistent with the Mstnb sequences in the Nile tilapia genome . Subsequently, a preliminary screening was carried out with the principle of conservation, and, finally, 32 miRNAs with high scores were selected to conduct different experiments. The Mstnb 3′ UTR was subcloned into the psiCHECK2 reporter plasmid and used in a dual luciferase reporter experiment. The results showed that miR-30a-3p, miR-181a-5p, and miR-181b-5p downregulated the relative Renilla/Firefly luciferase ratio (Rluc/Fluc) of the Mstnb 3′ UTR (P < 0.001) (P < 0.01) (P < 0.05)  ; miR-338 < 0.01) ; and miR < 0.05) . Special < 0.05) . Among t < 0.05) . Thus, mTo further verify the miR-181 expression pattern, we attempted to clone the miR-181 family, but the results showed that only miR-181b-5p was cloned in tilapia . TherefoMstnb 3′ UTR at 307–313 nt and 1022–1029 nt  were present in the muscle, testes, eye, brain, and spleen  was used to digest the muscle of tilapia. The research in fish cell culture developed quickly following Wolf establishing the RTG-2 cell line in rainbow trout  also induced muscle hyperplasia and body weight increase in zebrafish (mstnb-deficient zebrafish increased after 80 days post-fertilization (Myf5, MyoD, and MyoG was significantly increased after rainbow trout primary myosatellite cells were treated with MSTN-1 (mstnb participates in the regulation of muscle growth in tilapia in a similar way in fishes as in mammals.Since MSTN is a negative regulator, inhibition or mutation of this gene may be useful for muscle development . For ins in mice , and knoebrafish . In theilization . Similarlization . The mRNh MSTN-1 , which wh MSTN-1 , while ih MSTN-1 . It is hmyostatin b in tilapia.In this study, miR-181b-5p overexpression inhibited MSTNb and activated downstream gene expression. Our results suggest that miR-181b-5p may regulate the muscle growth of tilapia by targeting The datasets generated for this study are available on request to the corresponding author.The Tilapia used in this study were obtained from the local farm of Guangdong Tilapia Breeding Farm, Guangzhou, China. No specific permissions are required for the buying of fish. All animal experiments were performed with the approval of the Sun Yat-Sen University Animal Care and Use Committee and in full compliance with its ethical guidelines.WL and ZZ conceived and designed the experiments. ZZ, XY, JJ, and GY performed the experiments. ZZ and WL analyzed the data. ZZ and CS wrote the paper. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Increased intake of omega-6 rich plant oils such as soybean and corn oil over the past few decades has inadvertently tripled the amount of n-6 linoleic acid  in the diet. Although LA is nutritionally “essential”, very little is known about how it affects the brain when present in excess. This review provides an overview on the metabolism of LA by the brain and the effects of excess dietary LA intake on brain function. Pre-clinical evidence suggests that excess dietary LA increases the brain’s vulnerability to inflammation and likely acts via its oxidized metabolites. In humans, excess maternal LA intake has been linked to atypical neurodevelopment, but underlying mechanisms are unknown. It is concluded that excess dietary LA may adversely affect the brain. The potential neuroprotective role of reducing dietary LA merits clinical evaluation in future studies. Contrary to common misconception, the intake of LA’s elongation-desaturation product, arachidonic acid , and the essential n-3 fatty acid, alpha-linolenic acid , and its elongation-desaturation products eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has remained relatively constant at <1% energy since the early 1900s.6 Despite being a major part of the diet, LA has been considered non-functional in the brain because of its low concentration  compared to palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1n-9), DHA, and AA which make up over 84% of brain fatty acids in rats and humans.8Surprisingly, little is known about how this increase in dietary LA affects the brain. It is mainly viewed as an essential precursor to AA, which is important for neurodevelopment and other physiological processes.10 However, unlike AA and DHA which mostly incorporate into brain membrane phospholipids, the majority (~59%) of LA entering the brain is rapidly β-oxidized into aqueous products, likely composed of carbon dioxide, acetate, and other polar metabolites of β-oxidation.9 Some of the 2-carbon acetate molecules produced by β-oxidation are recycled into cholesterol via de novo lipid synthesis pathways within the brain.11LA is known to enter the brain at a rate of ~4 pmol/g/s, which is comparable to the rate of entry of AA, DHA, and other fatty acids into the brain.15 OXLAMs are lipid mediators known to regulate pain and inflammatory signaling in peripheral tissue,19 where they are abundant.20 In the brain, they are presumed to be formed by LOX, COX, CYP450, and sEH enzymes, but their role there is not fully understood.LA is also a precursor to oxidized products known as ‘oxidized linoleic acid metabolites’ (OXLAMs) that are produced by auto-oxidation or enzymatically via lipoxygenase (LOX), cyclooxygenase (COX), cytochrome P450 (CYP450), and soluble epoxide hydrolase (sEH).This review highlights the current state of knowledge on the role and metabolism of LA and OXLAMs in the brain. Studies linking LA and OXLAMs to reported biochemical, neuropathological, or behavioral endpoints in chickens, rodents, and humans are discussed, and directions for future research are proposed.25 a neurodegenerative condition characterized by necrosis, edema, and microvasculature abnormalities that can lead to ataxia and death.26 Table Studies in the late 1950s and 1970s showed that chicks fed a vitamin (V)-E deficient diet containing LA developed encephalomalacia,21 In a follow-up study, the authors found that a V-E deficient diet containing 1.5% ethyl-AA induced encephalomalacia faster than a V-E deficient diet containing 1.5% ethyl-LA or lard (mixed source of LA and AA).24 These studies demonstrated that when V-E is absent from the diet, AA and to a lesser extent LA, damage the brain of young chicks. ALA caused no damage.Dam et al. reported that 2-day-old chicks fed a V-E deficient diet containing 1.5% ethyl-LA (weight percent) from the 2nd day of life onwards, died from encephalomalacia within 28 days (11 out of 12 chicks), compared to chicks fed a V-E deficient diet containing ethyl-ALA (0 out of 12) or a V-E deficient basal diet lacking both LA and ALA (0 out of 12).23 Chicks from each maternal group were then fed a V-E free diet containing 4% or 10% oxidized safflower oil high in LA (~70%) for 31 days to induce encephalomalacia. Oxidation was achieved by heating the oil at 145 °C for 24 h.Follow-up studies in chickens demonstrated that oxidized LA metabolites  were the likely cause of encephalomalacia, and that this effect was linked by maternal dietary LA content. Bartov and Bornstein reported that maternal intake of a 4% soybean oil (relatively high in LA) or 4% tallow (low in LA) diet containing 5 or 25 mg/kg α-tocopherol acetate each, from 6 to 11 months of age, reduced egg-yolk LA concentration in the tallow group (independent of V-E dietary content), and decreased V-E concentration in yolk of both groups fed the tallow and soybean diets containing low α-tocopherol acetate (5 mg/kg).23 Hence, a high LA maternal diet low in V-E accelerated the development of ataxia and encephalomalacia-related mortality in chick offspring maintained on a V-E free diet containing oxidized safflower oil. Lowering maternal dietary LA or supplementing it with V-E was protective.The authors reported that regardless of safflower oil content of the chick diet (4 or 10% level), chicks born to hens that were fed the 4% soybean oil diet containing low (5 mg/kg) α-tocopherol acetate levels experienced a significantly higher frequency of ataxia and mortality compared to chicks born hens fed 4% soybean oil containing 25 mg/kg α-tocopherol, or beef tallow containing either 5 or 25 mg/kg α-tocopherol.25 They fed a V-E free diet containing oxidized safflower oil or non-heated safflower oil methyl esters at 4% or 10% (w/w) to chicks for 2 to 3 weeks. At 4% oil content, the incidence of ataxia and mortality were higher in the group fed the non-heated safflower oil compared to the group fed oxidized safflower oil. At 10%, ataxia and mortality were higher in the group fed oxidized safflower compared to the group fed non-heated safflower oil. The findings suggest that in the absence of V-E, oxidized safflower oil at 10% (w/w) induced more neurological deficits than at 4%. Also, the fact that ataxia and death were observed in the group fed 4% non-heated safflower oil, suggests the absence of dietary V-E might induce oil auto-oxidation of LA (the main PUFA in safflower oil). It is not known whether the OXLAM species generated by auto-oxidation due to the absence of V-E in the diet, differ from those generated by heating. Notably, this study did not include a control group provided with V-E in the diet.Budowski et al. performed a series of experiments to understand the cause of ataxia and mortaility linked to encephalomalacia.25 Ataxia and mortality were relatively high in the groups fed non-heated oils lacking or containing V-E . In the groups provided with oxidized oil with or without V-E, the incidence of ataxia and mortality were much increased , irrespective of V-E content. It is possible that higher doses of V-E might have been more effective in reducing mortality and the incidence of ataxia by limiting auto-oxidation, particularly in the non-heated safflower oil group.To test whether V-E was protective, the authors provided 1-day-old male chicks with non-heated or heated 10% (w/w) safflower oil methyl esters lacking or containing 1 µg/kg V-E for 19 days.25 Because oleic acid and LA are the main unsaturated fatty acids in safflower oil, the authors introduced oleic acid and LA derived ketone esters  at 0.2% and 0.12%, respectively, to the same V-E free diets containing 4% safflower oil. They found that the incidence of ataxia was 50% and 80% with the oleic acid and LA keto-esters, respectively, and thus higher than the diets lacking these oxidized compounds. A separate study by Kokatnur et al. showed that the addition of 0.25% (w/w) 12-oxo-cos-9-octadecenoic acid, 12-oxo-octadecanoic acid, 12-oxo-trans-10-octadecenoic acid, or 12-oxo-cos-9-octadecenoic acid methyl ester to a V-E deficient basal diet containing 10% corn oil, induced encephalomalacia in 42–100% of chickens compared to 17% in those fed the basal diet.27 The authors also determined that as little as 0.05% of ketone fatty acids induced encephalomalacia and that this effect was exacerbated by adding more corn oil to the diet (from 2.5 to 10% w/w).27 These studies provide direct evidence that OXLAMs, and to a lesser extent oxidized oleic acid metabolites, induce ataxia and mortality due to encephalomalacia in chickens.The authors also introduced the polar fraction (at 0.3% w/w) to the V-E free diet containing 4% safflower oil, and found that it induced ataxia and increased mortality compared to the same background diet lacking the polar extracts.25 suggesting that the neurotoxic effects of OXLAMs may be caused by blood coagulation and disturbances in the brain’s microvasculature. This is in agreement with a study by Fischer et al. who showed morphologic changes in cerebral vasculature, characteristic of pre-ceroid formation in adult chickens fed an 8% LA diet lacking V-E, compared to chickens on a similar diet containing V-E.22 Fully developed ceroids have been linked to neurodegeneration.28In the Budowski et al. study, the administration of the anti-coagulant dicumarol to a V-E free diet containing 4% safflower oil reduced the incidence of ataxia and mortality,The few studies that explored the direct role of LA and its metabolites in rodents have shown that LA is involved in regulating brain immunity, seizure threshold, and neuronal signaling, and that some of these effects are mediated by its oxidized metabolites .2 concentration and COX-2 activity induced by 2-day lipopolysaccharide (LPS) administration into the 4th ventricle.29 Two independent studies reported that dietary LA lowering (from 5.2% to 0.4% energy) reduced brain concentrations of pro-inflammatory AA-derived eicosanoids  and increased concentrations of anti-inflammatory EPA or DHA derived metabolites.31 This may partially explain why LA lowering increased resilience to LPS-induced neuroinflammation in the Taha et al. study.29In rats, LA lowering was found to attenuate LPS-induced neuroinflammation, reduce brain concentration of pro-inflammatory lipid autacoids and increase brain levels of anti-inflammatory lipid mediators. Taha et al. reported that compared to a “high” 5.2% energy LA diet, chronic consumption of a “low” 0.4% energy LA diet for 15–18 weeks, reduced the incorporation rate of radiolabeled AA into the brain and prevented the increase in brain AA-derived prostaglandin E32 Voskuyl et al. showed that intravenous administration of 56–64 mg/kg LA raised cortical focal and generalized seizure thresholds by 3-fold and 1.3–9.0-fold, respectively, in a cortical stimulation seizure model compared to saline and oleic acid controls.33 The effect of chronic LA administration on seizure thresholds has not been tested.Acute administration of LA was reported to confer seizure protection in rats. When infused intravenously at a dose of 0.028 mg/kg, it decreased the incidence of tonic-clonic convulsions by 3-fold, but increased the incidence of cortical spike-wave discharges by 1.5 to 2 folds.34 Direct application of LA itself to rat hippocampal slices did not alter paired-pulse facilitation within soma or dendrites, whereas LA-derived 13-hydroxyoxtadecadieonic acid (13-HODE) increased somatic paired pulse facilitation.34 Although the Hennebelle et al. study raises the possibility that LA’s effects on the brain might be mediated by OXLAMs,34 it only tested one OXLAM (13-HODE). Future studies should address the isolated and collective effects of other hydrox, epoxy, dihydroxy, trihydroxy, and ketone metabolites of LA on neurotransmission and other brain processes.Recent studies suggest that the effects of LA in the brain are likely mediated by OXLAMs. Hennebelle et al. showed that OXLAMs are increased in various rat brain regions following acute ischemic injury, possibly due to their involvement in the response to ischemic brain injury.34 it is not known whether they are preferentially formed in the brain from LA, or incorporated directly from circulating OXLAMs provided in the diet. This issue was recently addressed by two studies. Taha et al. fed rats a graded LA diet  for 15–18 weeks, and showed that increasing dietary LA increased cerebral cortex and cerebellum OXLAM concentrations in a dose-dependent manner , or a low LA diet (4% energy) with or without oxidized corn oil as a source of OXLAMs.35 The authors found no evidence of dietary OXLAM incorporation into the brain, but consistent with a previous report, the high LA diet increased brain OXLAM concentrations compared to the low LA diet with or without OXLAMs.35 These studies suggest that dietary OXLAMs are not readily incorporated into rodent (mouse) brain, and that LA is the main source of OXLAMs in the brain. Behavioral studies on dietary OXLAMs in rodents are yet to be performed, as has been done previously in chickens. Notably, although the chicken studies outlined in Table 36Although OXLAMs are known to be abundant in rodent brain,37 Although it is difficult to establish causation with such studies, the same study, found no association between LA on neurodegenerative disorders after adjusting for confounders using multi-variate analysis, but reported a significant 4–15% risk reduction in neurodegeneration with increased AA, ALA, or EPA and DHA intake.37 Randomized controlled trials are needed to confirm these findings.Most of the clinical evidence on the effects of LA on the brain stem from epidemiological studies. One prospective cohort study reported an inverse association between high PUFA intake and mortality linked to neurodegenerative disease.39 Lowering dietary LA without changing EPA and DHA did not significantly alter migraine frequency or improve quality of life.39 Although this study did not have a parallel group that received EPA and DHA alone, it provides evidence of a potential synergistic benefit to lowering LA and increasing EPA and DHA simultaneously.40 It is possible that longer-term LA lowering regimens might reduce migraine frequency as well, although this remains to be tested.Ramsden et al. reported that dietary LA lowering from 7% to 2% energy combined with a high EPA and DHA diet (~1.5 g per day) for 12 weeks, reduced migraine frequency and improved quality of life in patients with drug-resistant migraines.41 Thus, consistent with the increase in dietary LA in the food supply, breastmilk LA composition has increased from 7% to 12% of total fatty acids between 1970 and 2000.42 The 12% composition value corresponds to 8% energy, which exceeds the minimum 1–2% energy required for developing infants by 4–8-fold.2Several epidemiological studies explored the role of LA on neurodevelopment. Like other dietary fatty acids, LA accumulates in breast milk, which is the primary source of infant nutrition for the first few months months of life.43 In the same cohort, maternal breastmilk LA percent composition was associated with reduced verbal IQ at 5 to 6 years of age.44 In fact, children breastfed with the highest levels of LA had cognitive scores comparable to children who were never breastfed.43 These effects were independent of AA or DHA breast milk composition, suggesting a direct impact of excess breast milk LA  on brain development. Consistent with these findings, Lassek and Gaulin found an inverse correlation between breast milk LA percent composition and cognitive scores in 15-year-old children, suggesting a long-lasting impact of maternal LA on offspring cognitive skills.45 Steenweg-de Graaff et al. also reported a significant positive association between maternal plasma LA composition measured at mid-pregnancy and the risk of autistic traits in children at the age of 6 years.46 A more recent study found that prenatal intake of diets high in the ratio of LA to ALA, was associated with a 2-fold increase in the risk of delayed psychomotor and mental developmental in 6 months infants.47Overall, studies have shown an adverse effect of maternal breast milk or dietary LA on neurodevelopment. One study reported that a high maternal breast milk LA percent composition (>9.7% of fatty acids) was associated with reduced motor and cognitive scores in 2- to 3-year-old infants.Pre-clinical and clinical studies dispel previous assumptions that LA is a benign fatty acid in the brain. On the contrary, when present in excess and chronically, it induces ataxia in chickens, promotes neuroinflammation in rats and is linked to abnormal neurodevelopment in humans. LA administered acutely to rats confers seizure protection, although its chronic effects on seizures has not been tested.48 the extent of their bioavailability and accumulation in the brain from dietary sources appears to be minimal.36 However, further assessment of their degradation products on brain neurophysiology and other organs is warranted.Recent animal studies suggest that the effects of LA might be mediated by OXLAMs generated from LA entering the brain. Although OXLAMs are present in the diet,31 and human studies showing that LA lowering combined with EPA and DHA reduced headache frequency in patients with drug-resistant migraines.39Chronic consumption of low LA diets might protect the brain against inflammation, as evidenced by rat studies showing an anti-inflammatory lipidome in the brains of rats fed a low LA diet,In conclusion, this review presents evidence that excess LA in the food supply might adversely affect the brain. The potential benefit of LA lowering merits detailed evaluation in well-designed and adequately-powered clinical studies, to test whether this translates into tangible reductions in the risk of neurodegenerative disorders and neurodevelopmental abnormalities at a population level.figure permission


The astroglial response was also explored by co-immunostaining for GFAP and S100b together with p-JNK and it was found to be particularly exacerbated in the MPTP+NAC+HA-1077 group. The unexpected toxic effects found in the combined use of NAC and HA-1077 in old-Parkinsonian mice highlight the importance of taking into account that in elderly Parkinsonian patients the combination of some drugs  can have side effects that may result in the exacerbation of the neurodegenerative process.The design of therapeutic strategies that focus on the repositioning of anti-inflammatory and antioxidant drugs are a great bet to slow down the progression of neurodegenerative disorders. Despite the fact that Parkinson’s disease (PD) is an age-related pathology, almost all experimental studies are carried out in young animals. Here, we evaluated the possible neuroprotective effect of the combination of the antioxidant N-acetylcysteine (NAC) and the anti-inflammatory HA-1077 in aged 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice , whose individual treatment has been shown to have neuroprotective effects in this Parkinsonism model. Interestingly, NAC+HA-1077-based treatment produced a significant increase in dopaminergic neuronal death accompanied by an increase in microglial and astroglial activation in the Substantia Nigra  This fact implies the development of motor alterations as stiffness, bradykinesia, resting tremor and postural instability. PD is the second most prevalent neurodegenerative disorder that affects 100–200 per 100,000 people at the age of 65–70 years (Parkinson’s disease (PD) is a pathology based on the chronic and the progressive loss of dopaminergic neurons in the Substantia Nigra 70 years . Althoug70 years  over oth70 years  or expos70 years .post-mortem analysis have been carried out in PD patients with genetic predisposition along with age-associated immunological alterations that have demonstrated an association between slight inflammation in the CNS and vulnerability to the degeneration of dopaminergic neurons  and antioxidant  in old-Parkinsonian mice. Both drugs have been shown to have an individual beneficial effect on both dopaminergic neuronal death and glial response. HA-1077 is a ROC-kinase inhibitor, a protein that is essential for the motility and phagocytic capacity of microglia. Previous studies from our research group showed that the use of HA-1077 decreases the activation of microglia in MPTP-intoxicated mice . Additioad libitum, under ambient conditions (20 ± 2°C) and light cycles: darkness of 12:12 h. All animal testing methods and procedures were approved by the Council of the European Community Committee (2010/63/EU) and by the Institutional Committee on Animal Ethics of the University of Murcia (REGA ES300305440012).The studies were performed on 64 male elderly C57BL/6J mice  provided by Eleverge Janvier  and maintained in an isolated room with access to water and food n = 24) and MPTP group (n = 40). Parkinsonism was induced via intraperitoneal (i.p.) injection of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine , which specifically causes dopaminergic neuronal death. The dose administered per animal was 30 mg/kg, divided into two injections of 15 mg/kg each and spaced 5 h over a day  were divided into four subgroups according to the treatment: (i) control (untreated); (ii) NAC; (iii) HA-1077; (iv) NAC+HA-1077. NAC treated groups received a dose of 100 mg/Kg (i.p.) of NAC (Sigma Aldrich) half an hour after each injection of MPTP (NAC and MPTP+NAC and MPTP+NAC+HA-1077), following the protocol described by Seventy two hours after the drug treatment, the animals were sacrificed by cervical dislocation under an overdose of ketamine  and Xylazine . For immunohistochemistry and immunofluorescence, brains were extracted, fixed overnight at 4°C in 4% paraformaldehyde in phosphate buffered saline or PBS , washed with ethanol and embedded in paraffin. For Western Blot and ELISA, brains were extracted and immediately frozen. The cerebellum, midbrain and striatum were dissected according to the coordinates of the mouse brain atlas . The tisn = 3 in the No-MPTP groups  and n = 5 in the MPTP groups . For Western Blot and ELISA experiment, the number of animals used was n = 3 in the No-MPTP groups  and n = 5 in the MPTP groups .The number of animals used for immunohistochemical (IHQ)/immunofluorescence (IF) analysis was 2O2 for 20 min and non-specific binding of the antibodies was blocked (both in immunohistochemistry and immunofluorescence) in a blocking solution for 30 min: tris-buffered saline 0.1 M pH 8.4 (TBSIHQ), 10% goat serum and 0.5% Triton X-100. After two washes with TBSIHQ the samples were incubated with the corresponding primary antibody overnight at 4°C (Table IHQ+Tween 0.5%+1% goat serum at the dilution indicated in Table IHQ and sections were incubated with the secondary antibody diluted in TBSIHQ at the concentrations indicated in Table IHQ and incubated at room temperature (RT) with avidin/biotin conjugated to peroxidase , staining using the kit 3,3′-diaminobenzidine  and, finally, assembly was performed for observation under the optical microscope. In the case of immunofluorescence, after washing with TBSIHQ after incubation with the secondary antibody, the samples were assayed .Coronal sections (7 μm) of SNpc and striatum were cut on microtome . The sections were deparaffinized in xylene, rehydrated in a gradient of ethanol  and distilled water. Antigenic retrieval was performed in citrate buffer 30 min at 95°C . In immunohistochemistry, endogenous peroxidase was inhibited with a solution of 0.3% HFor DAB labeling quantification, images were obtained by Hall 100 ZEISS optical microscope with an Axiocam ZEISS digital camera and were analyzed using the NIH ImageJ software . Field size was set at 1088 × 1040 pixels. All the analysis were performed without post-processing the images.SNpc and striatum areas were delimitated according to anatomical coordinates . For eac3. Counting unit was the nucleus surrounded by immunoreactive cytoplasm. The area of the SNpc was calculated by a systematic non-biased method. In the striatum, the dopaminergic innervations were quantified by measuring the optical density (O.D.) of DAB signal, expressed as Area (%)/mean gray value  with the 63× glycol-immersion objective lens at optical zoom of 0.75 . In order to avoid quantification and measurement bias, all the images were obtained under the same setting conditions. Morphological analysis was performed by O.D. measure following the protocol described by WB) + 0.05% Tween-20 (TBST) + 5% BSA. Membranes were then incubated with the specific primary antibody at 4°C overnight and constant stirring  from each tissue extract were resolved in 12% polyacrylamide gels under denaturing conditions at constant voltage (110 V) at RT. After electrophoresis, proteins were transferred to PVDF membranes  at constant voltage  using the transfer buffer: 25 mM Tris-HCl, 192 mM glycine, 0.1% SDS, 20% methanol. Membranes were incubated for 1 h and constant stirring in blocking solution: 0.1 M TBS pH 7.5  according to the manufacturer’s protocols. Signal generated was read as absorbance at 450 nm using CLARIOstar reader (BMG LABTECH).Total JNK1/2 levels and phosphorylated-JNK1/2 levels were measured using Enzyme-Linked Immunoabsorbent Assay (ELISA). The assay was conducted using JNK1/2 (pT183/Y185) + Total JNK1/2 SimpleStep ELISApost hoc analysis Sidak. The null hypothesis was rejected considering values of p < 0.05 in all cases.Data are shown as mean ± SD. The statistical analysis were performed with GraphPrism 7.0 software using a two-way ANOVA test followed by p = 0.0026) compared to the control group. These results validated our model for Parkinsonism. Similar results were obtained for the dopaminergic cell loss of the MPTP+HA-1077 group compared to its control (p = 0.0025). Surprisingly, no significant differences were observed in the TH+cells/mm3 in the MPTP+NAC+HA-1077 group compared to the control group  and MPTP+NAC+HA-1077 (p = 0.0117) groups compared to their respective No-MPTP groups . These results demonstrate for the first time that NAC has a neuroprotective effect on dopaminergic neuronal death in old mice after MPTP insult, opening the action spectrum described by To verify the possible neuroprotective effect of the combined treatment NAC+HA-1077 on the loss of dopaminergic neurons, immunohistochemical staining of TH in SNpc and in the striatum was performed Figure . As showp Figure . The levs Figure . In contp = 0.002) and MPTP+NAC+HA-1077 (p < 0.0001), compared to their respective No-MPTP groups  and MPTP+NAC+HA-1077 (p < 0.0001) groups compared to their respective control groups. The number of Iba-1+ cells of MPTP+NAC and MPTP+HA-1077 groups in the striatum was not significantly increased compared to their control groups. These results support those obtained in relation to dopaminergic neuronal death and highlight the anti-inflammatory effect of individual treatment with NAC or with HA-1077. In contrast, Iba-1+ cells in the MPTP+NAC group was significantly lower compared to the NAC No-MPTP group (p = 0.0011).In the striatum Figure , there wp = 0.0215) and MPTP+NAC+HA-1077 (p = 0.0150) groups compared to their control animals , MPTP+HA-1077 (p = 0.0009), and MPTP+NAC+HA-1077 (p = 0.0217) groups compared with their No-MPTP groups. The astrocytes’ morphological analysis in the SNpc showed a slight increase of Mean O.D. value of the GFAP immunostaining in the MPTP, MPTP+HA-1077 and MPTP+NAC+HA-1077 groups compared to their respective No-MPTP groups in the SNpc  and very significantly increased in MPTP+NAC+HA-1077 (p < 0.0001) compared with their respective control groups . The study of morphological changes of astrocytes in the SNpc showed significant changes in MPTP+HA-1077 (p = 0.0208) and MPTP+NAC+HA-1077 (p = 0.0249) compared with their No-MPTP groups. Moreover, in the striatum showed that mean O.D. value was significantly increased in MPTP+NAC+HA-1077 (p = 0.0002) group compared to its No-MPTP group  and MPTP group treated with HA-1077 (p = 0.0129) compared to their respective controls. Furthermore, a significant increase in p-JNK levels was observed in the MPTP+HA-1077 group (p = 0.0007) and, reinforcing previous results, in the MPTP+NAC+HA-1077 group (p = 0.0219).Then, quantification of total JNK and p-JNK levels was performed by ELISA assay in the midbrain extracts Figures . The resIn addition, immunofluorescence staining for GFAP and S100b with p-JNK was performed at the SNpc level Figure  in orderpost-mortem studies, we first analyzed dopaminergic neuronal death by TH immunostaining in the SNpc and the striatum  may be MAPKinases pathway . MAPKinaAccording to the results obtained in the present study, we postulate that the combination of HA-1077 and NAC administrated in the most vulnerable brains, because of aging, has turned out to increase neuroinflammatory processes. With this, we conclude that personalized therapy should be supported in patients with different treatments based on the described data. Therefore, this work opens the door to consider the study of the effects of the combination of common drugs on neuronal death and inflammation, in order to design therapeutic strategies more adapted to PD patients (mainly in elderly people).ALG-M and LC carried out all the experiments and wrote the manuscript. CS-R, CE, and EF-V participated in the experiments. MTH conceived the idea and design of the study, discussed the results, and worked in the manuscript preparation. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this assay, a short single-stranded 76-mer DNA aptamer with a unique DNA sequence, which binds towards the cyto-c like an antibody with a high binding affinity and specificity, was conjugated to gold nanorods (AuNR) for LSPR sensing. Practically, cyto-c was first grabbed by a capturing antibody functionalized on the surface of micro-magnetic particles (MMPs). Subsequently, the AuNR-conjugated aptamer was added to form a complex sandwich structure with cyto-c -(cyto-c)-(AuNR-aptamer)) after washing away the non-target impurities, such as serum residues and intracellular contents, in a microfluidic chip. The sandwich complex led to formation of AuNR aggregates, which changed the LSPR signals in relation to the amount of cyto-c. With the LSPR signal enhancement effects from the AuNRs, the detection limit of cyto-c, sparked in human serum or culture medium, was found to be 0.1 ng/mL in our platform and the whole sensing process could be completed within two hours. Moreover, we have applied this assay to monitor the apoptosis in leukemia cancer cells induced by a potential anti-cancer agent phenylarsine oxide.To determine the degree of cancer cell killing after treatment with chemotherapeutic drugs, we have developed a sensitive platform using localized surface plasmon resonance (LSPR) and aptamers to detect the extracellular cytochrome- In cancer therapy, apoptosis is the primary process that eliminates cancer cells after drug treatment and the release of cyto-c from the mitochondria into the cytoplasm is an essential step and is regarded as a ‘point-of-no-return’ stage of apoptosis. Indeed, the serum level of cyto-c increased at the onset of chemotherapy, reached the peak with a median of two days, and decreased gradually during remission induction [c found in the blood circulation correlates to the percentage of cancer cell death, i.e., the efficacy of the anti-cancer drug treatment [Cytochrome- therapy ,2. Durinnditions ,4. SerumRS/MODS) ,6,7,8. Induction ,10. Therreatment ,12.c selectively, and it also acts as both the recognition element and the bio-barcode reporter in the ABC assays [c with high affinity and high specificity in a way similar to that in the antibody-antigen reactions. Due to its unique DNA sequence, it can serve as a simple bio-barcode reporter in the ABC system for the sandwich complexes ((micro-magnetic particles (MMP)-capturing antibody (Ab))-target-(aptamer)) after DNA amplification. These ABC assays have been introduced previously to sensitively and rapidly screen the cell death marker in multidrug resistant cancers as part of the drug screening process [Previously, we have developed a number of aptamer-based bio-barcode (ABC) assays, in which a 76-mer DNA aptamer was employed to capture cyto-C assays ,14. This process ,15. In t process ,16. TherNanomaterial-based localized surface plasmon resonance (LSPR), which employs a disconnected pattern of the metallic surface on nanomaterials to generate a non-propagating plasmon, has been drawing much attention these days because the LSPR platform provides several advantages over other optical label-free techniques. First, different functionalized nanomaterials coated with recognition elements can be applied in a solid or a solution format for high-throughput target detection . Second,c sensing in human serum on a microfluidic platform using a label-free LSPR approach without DNA amplification. As shown in c bound by the capturing Ab on MMP. Mimicking the clinical situations during chemotherapy, cyto-c of the indicated concentrations were sparked in human serum. Results from our study confirmed that this LSPR-AuNR assay is a convenient and effective approach for determining the concentration of cyto-c in the serum. With the growing demand on the healthcare market, especially the need of a convenient, user-friendly, and sensitive point-of-care (POC) biosensor for bedside utility, it is likely that our assay in the microfluidic platform will go beyond academic communities to be a practical POC biosensor for clinical diagnosis and prognosis.On the other hand, microfluidics adds another dimension to enhance the robustness of detection. Microfluidic chips provide a micron-scale fluidic actuation with advantages over the current benchtop method by reducing the volume and the size of liquid handling. Additionally, sequential fluidic flow of the assay can be controlled by fluidic actuation of different channels of reagents . Therefoc biotinylated-aptamer was 5′-biotin-TEG-ATCGATAAGCTTCCAGAGCCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGCCGTAGAATTCCTGCAGCC-3′, where the italics represent the constant region of our aptamer during the systematic evolution of ligands by exponential enrichment (SELEX) process in our previous reports [c Ab (sc-7159) was obtained from Santa Cruz Bio-technology . Recombinant streptavidin was obtained from Life Technologies . Cyto-c, bovine serum albumin (BSA), phenylarsine oxide (PAO), and other reagents were purchased from Sigma-Aldrich. Fetal bovine serum and horse serum were obtained from Life Technologies. Human blood was obtained from healthy donors by puncturing fingertips with sterile lancets with depth settings with informed consent and approval by the University Clinical Research Ethics Committee (CRE-2012.025). To obtain the human serum, the non-heparinized blood was centrifuged at 2000× g for 10 min and the supernatant was collected. All sera were diluted to 10% (v/v) with sterile phosphate buffered saline (PBS) before use.Aptamer was synthesized by Integrated DNA Technologies. The sequence of the 76-mer cyto- reports ,15. Biot reports . MMP Pro630 nm = 0.2) and the mixture was incubated overnight at room temperature. The PEG-displaced AuNRs were washed twice with DNase-free deionized water using centrifugation at 8000× g for 10 min. N-ethyl-N′-(3-(dimethylamino) propyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) chemistry was conducted to activate the carboxyl group of HS-PEG-COOH for amine group attachment. Briefly, 0.4 M EDC and 0.15 M (NHS) were prepared freshly and mixed in a 1:1 ratio. The mixture was then loaded to the PEG-displaced AuNRs and incubated for 15 min at room temperature. The ECD/NHS was removed by centrifugation and streptavidin (100 μg/mL) was then added and incubated for 30 min. To visualize AuNR targeting the MMP-Ab-cyto-c complex by confocal microscopy , enhanced green fluorescent protein (EGFP) (100 μg/mL) was added with streptavidin during protein binding on the AuNRs’ surface. Blocking agent ethanolamine (10 mM) was added to the mixture for 10 min to block all activated carboxyl groups. Biotinylated-aptamer (10 pmol) was then added and incubated for another 30 min. The AuNR-aptamers were washed twice with DNase-free deionized water and ready for the downstream AuNR-LSPR assay. AuNR (length:width = 47.3 ± 2.4:22.3 ± 1.6 nm) were synthesized as described in our previous publication and stabilized with cationic surfactant cethyltrimetylammonium bromide (CTAB) ,26. To rTo validate the proper functionalization of AuNR-aptamers, AuNRs from each sequential step of functionalization were mixed with SYPRO protein  dye for protein quantification or SYBR safe  DNA binding dye for DNA quantification in a 96-well plate and incubated for 30 min at room temperature, prior to the fluorescence detection with excitation/emission 485/625 nm and 485/540 nm for protein and DNA measurements, respectively. BSA of standard concentrations from 0 μg/mL to 100 μg/mL was used to calculate the protein conjugated on the AuNRs. Aptamer DNA of standard concentrations from 0 nM to 1000 nM was used to calculate the aptamer conjugated on the AuNRs.The disposable microfluidic chamber, a polydimethylsiloxane (PDMS)-based polymer, of length × width × depth of 20 × 5 × 1 mm, was replicated from a poly(methyl methacrylate) (PMMA) mold fabricated with computer numerical control milling. Liquid PDMS was poured onto the PMMA mold and incubated at 60 °C for 2 h. The bottom of the solidified PDMS microfluidic chip was then sealed with a transparent adhesive purchased from Life Technologies. The inlet and outlet were connected with the tip of a micropipette to guide the fluid A.c Ab at 25 °C for 1 h and then blocked with 1% BSA in the binding buffer  at 25 °C for 1 h. The MMP-Ab was then loaded into the microfluidic chamber with permanent magnet placed at the bottom to hold the MMP-Ab -1640 medium  with 20% (v/v) fetal bovine serum (FBS). The standard cyto-c solutions or samples were loaded into the microfluidic chamber and incubated at 25 °C for 1 h. After that, the MMP were then washed once with 1 mL binding buffer plus 0.1% tween-20 and then with 1 mL DNase-free deionized water at a flow rate of 500 μL/min. Fifty microliters AuNR-aptamer was then added into the microfluidic chamber without magnet holding, and the complex was added into a 96-well plate for downstream detection by scanning the absorbance from 400 nm to 1000 nm with an absorbance reader .The MMP was conjugated with mouse anti-cyto-e MMP-Ab A. Cyto-c5/mL HL60 cell lysate, Santa Cruz Biotechnology, Dallas, TX, USA) samples were used. A benchtop washing method was followed in previous studies, except a one-time washing with 1 mL buffer, i.e., the same volume as that of the microfluidic chip method, for a fair comparison [To evaluate the effectiveness of impurities removal by the assay using the microfluidic chip, compared with benchtop method on effectiveness of impurities removal, serum (10% FBS) and cell extract (1 × 10mparison . Proteinv/v) FBS were collected, washed twice with PBS, and resuspended to 1 × 105/mL in serum and treated with PAO of various concentrations or positive control (digitonin (5 μM)) were loaded and incubated at 37 °C for 1 h. Cell-free serum was collected by passing the sample through a 0.2 μm filter unit. The serum was then subjected to AuNR-LSPR assay. The cells were also subjected to Annexin-V-GFP  and Propidium iodide (PI) staining for flow cytometric analysis to determine the early and late apoptotic response. Briefly, the cells treated with PAO for one hour were stained with Annexin-V-GFP  for 10 min and PI  for 20 min in the dark. The cells were then washed twice with PBS. Subsequently, green and red fluorescence were detected with an excitation at 488 nm by a flow cytometer , Franklin Lakes, NJ, USA). The fluorescence properties of 1000 cells were collected for the analysis.Human acute promyelocytic leukemia HL-60 cells were obtained from ATCC . For studying the anti-cancer effect of PAO, HL-60 cells cultured in the RPMI medium with 20% . Results are mean ± standard error of the mean (SEM) from at least three independent assays. Statistical analysis of the experimental results was conducted by Student’s c sensing application, the optimization of aptamer conjugation on AuNRs was conducted to obtain the best functional nano-sensing units. PEG with sulphyl (-SH) group only (mPEG-SH), and PEG with both a sulfur group and carboxyl (-COOH) group (HS-PEG-COOH) were used to displace CTAB from AuNRs. Since only the latter provides the carboxyl group for further AuNR functionalization, the ratio of mPEG-SH to HS-PEG-COOH was adjusted to optimize the condition for AuNR functionalization without losing its stability due to CTAB removal. c sensing could be obtained.Prior to the cyto-After the optimization of PEG ratio on AuNR conjugation, the quality of PEG-functionalized AuNRs on downstream functionalization with streptavidin and then the target aptamer was evaluated. Protein binding fluorescence SYPRO red dye was used to evaluate the amount of bound streptavidin protein, and DNA binding fluorescence SYBR safe dye was used to evaluate the amount of bound aptamer DNA. As expected, the previous optimized mPEG-SH:HS-PEG-COOH in a 9:1 ratio yielded the highest binding percentage of streptavidin and aptamer, compared with other ratios C,D. Theoc standards of known concentration and the results were shown in c used in the AuNR-LSPR for the construction of a standard curve  + (3 × (0.006)) = 0.018 aggregation ratio unit for AuNR. The LOD of AuNRs was found below 0.1 ng/mL cyto-c from the standard curve. Standard concentration of cyto-c in culture medium was also used for a fair comparison of LOD with other studies that also used culture medium (c), suggesting the culture medium did not pose any negative effects on sensitive detection in this assay.The limit of detection (LOD) is defined by the International Union of Pure and Applied Chemistry (IUPAC) as the mean of the blank measured plus triple its standard deviation (SD) for a higher than 99% confidence level . The SD e medium B. Althouc. It was found that green fluorescence only appeared in MMP complex when cyto-c was present  as a tag. DNA binding dye was avoided in this case since the binding action of the dye on DNA double strands would affect the secondary structure of aptamer DNA, thus altering its binding ability on cyto- present C. To furc in serum, we need to understand if any unwanted signals, which affect the LOD due to non-specific binding were found in a negative control in the serum. A standard solution of known cyto-c concentration in sera from different sources  were prepared and used for cyto-c sensing. As expected, we did not see any significant difference of the cyto-c in sera when compared with that in buffer  in serum were treated with serum alone, digitonin (5 μM), or PAO  for 1 h at 37 °C, and 5% CO2. Cell-free sera from the treated and control groups were collected by filtering them with a 0.2 μm filter unit, and were subjected to the AuNR-LSPR assay. As expected, a low concentration of cyto-c (0.5 ± 0.1 ng/mL) was found in the untreated control, indicating that cancer cells were intact in the serum. On the contrary, cyto-c was released upon PAO treatment in a dose-dependent manner  and the digitonin-treated sample was found with a high concentration of cyto-c (>100 ng/mL). To verify the drug-induced apoptosis in the cells, Annexin-V-GFP, which targets the outer membrane leaflet during early apoptosis, and PI, which targets the nuclear DNA with a non-selective permeable or leaked plasma membrane during late apoptosis, were applied by cell staining for flow cytometric analysis. Obviously, we found only a small increase in green fluorescence in the lower right quadrant in the samples treated with PAO for one hour when compared with that of control  method that a simple absorbance reader is required to carry out signal measurement. Additionally, because of the strong LSPR effect in the AuNR-SPR assay, the detection limit on cyto-c reported in this study was better than the other techniques using enzymes for signal amplification.Indeed, we have developed a number of platforms using aptamers to determine the cyto-c concentration in patients with different diseases. For example, the serum concentration of cyto-c in patients with SIRS was increased up to 210 ng/mL compared with that of healthy subjects (<0.1 ng/mL) [c concentration was below 20 ng/mL, while that in the patients with malignant tumors and low survival rate (over 75% of the patients) was found to be over 40 ng/mL [c concentration in the above scenarios for clinical diagnosis. Additionally, the simple operation using LSPR absorbance change enables health providers, who are not trained for the medical laboratory techniques, to read the results easily. In fact, this AuNR-LSPR assay using the microfluidic format could extend to test other disease markers  easily with a simple replacement of the corresponding recognition elements. We expect that this detection platform will have a variety of uses in the coming future.There are reports showing the change in serum cyto-1 ng/mL) ,10. In t40 ng/mL . Obviousc sensing in human serum to monitor the efficacy of cancer treatment. This AuNR-LSPR platform is rapid, robust, and simple. In the presence of cyto-c, the AuNRs conjugated with cyto-c-specific aptamers will form aggregates, thus giving rise the LSPR effects. Practically, the LOD for cyto-c was found to be 0.1 ng/mL in the buffer, culture medium, and serum of different animals, including fetal bovine, horse, and human. As a proof of concept, we have successfully demonstrated the utility of this platform to detect the cyto-c released from leukemia cells upon anti-cancer drug PAO treatment. Taken together, our simple AuNR-LSPR detection platform is expected to have a wide range of potential for detecting disease markers in the future.We have demonstrated a new approach using a label-free LSPR format with AuNRs for cyto-


A new type of gluten-free pasta has been developed based on a rice-buckwheat mixture. The aim of the study was to investigate the effect of process parameters of moisture content , barrel temperature , and screw speed  on cooking and textural properties of rice-buckwheat pasta produced by a single-screw extrusion-cooker. The process uses response surface methodology based on a Box-Behnken experimental design. Results showed that with regard to this rice-buckwheat pasta, raising moisture content of the raw materials increased cooking loss and stickiness, but decreased firmness, while increasing barrel temperature reduced cooking loss and stickiness, but increased hardness and firmness. Screw speed increase also affected positively hardness and firmness of the obtained products. Thus, optimal conditions  were established to produce good quality rice-buckwheat pasta. At this optimum, the pasta showed a compact and homogeneous inside microstructure. Furthermore, the pasta products exhibited low cooking loss (less than 6%), good hardness and firmness, with low stickiness and acceptable scores for all sensory attributes and for overall quality. Pasta is universally consumed and appreciated due to its palatability, easy preparation, inexpensiveness, and long shelf-life . Durum wAt present, a strict life-long gluten-free diet is the only effective therapy for this disease. As a result, gluten-free products have been developed, not only for celiac patients and those with a gluten-related disorder but also for the rest of population . HoweverFagopyrum esculentum) has certain nutritional and functional properties which are deficient or absent in gluten-free cereal grains (rice and corn) [Among raw materials, buckwheat (nd corn) . The intnd corn) ,6,7. Duend corn) .The extrusion-cooking technique is one of the most appropriate technologies for the production of gluten-free pasta ,10. MoreHowever, to the authors’ knowledge, there are no studies about the production of gluten-free pasta based on rice and buckwheat flours by means of the extrusion-cooking technique. Therefore, we investigated the use of buckwheat flour to enrich rice-based pasta. The aims of this work were (a) to study the effect of extrusion-cooking parameters on cooking and textural properties of rice-buckwheat pasta, and (b) to evaluate the quality of pasta made using optimum parameters.Rice flour was obtained from Lubella Sp. z o. o. S.K. . Buckwheat flour was bought from a local market and was sieved to obtain flour with particles sizes below 0.5 mm.Gluten-free pasta was prepared using a mixture of rice flour and buckwheat flour with a ratio of 50:50. The blends were then moistened to a pre-determined level of moisture content  and allowed to stand for 30 min. A single screw extrusion-cooker type TS-45  equipped with an additional glycol cooling section before the die was used to produce pasta products at different barrel temperatures  in the first extruder section, 100 °C in the second section, and 70 °C in the cooling section. The pasta products were processed at 60, 80, and 100 rpm using the barrel configuration of length to screw diameter L/D = 18. Rice-buckwheat pasta products were shaped like spaghetti using a die with 12 × 0.80 mm round openings and cut for 30 cm pieces. After pre-drying for 5 min at room temperature, the pasta strands were distributed on perforated trays and then dried in an air dryer at 40 °C for 4 h. The dried pasta (moisture content below 12%) was cooled, packed in sealed plastic bags, and stored for further analysis.X1 moisture content, X2 barrel temperature, and X3 screw speed) on the cooking and textural properties of the produced rice-buckwheat pasta. This design is a three-factor, three-level design. Each of the independent variables was coded at three levels . These correspond to low, intermediate, and high levels, respectively. The variables were applied as follows: 30, 33, and 36% for moisture content, 80, 100, and 120°C for barrel temperature and 60, 80, and 100 rpm for screw speed, respectively. The total number of experiments came out to be 15, with three replications at the center point , hardness (Y2), firmness (Y3), and stickiness (Y4). The optimal pasta samples were examined further for chemical composition, water absorption capacity, expansion ration, color, sensory attributes, and microstructure. A second-order polynomial model (Equation (1)) was defined to fit the responses:Y is the response obtained by the fitted model and β0 is the model intercept. β1, β2, and β3 are the linear coefficients, β12, β13, and β23 are the interaction coefficients, and β11, β22, and β33 are the quadratic coefficients.Responses included cooking loss (Yi) into individual desirability functions (id) that vary from 0 (unacceptable response) to 1 (completely desirable response). The overall desirability (D) with n responses is calculated by applying the geometric mean (Equation (2)). Subsequently, an algorithm is applied to determine the levels of variables that maximize D [To obtain optimal parameters for the production of rice-buckwheat pasta, an optimization method was applied using a desirability function. This approach consists of standardizing and converting each response . Protein (AACC 46-10), lipid (AACC 30-10), and ash (AACC 08-01) content were determined in triplicate according to AACC methods . Dietaryv/v). The obtained extracts were stirred for 30 min using a rotator Multi RS-60 operating at 5000 rpm , then they were centrifuged (5000 rpm) for 10 min at 4 °C, using centrifuge MPW-352R . The extraction was performed in duplicate. The supernatants were recovered and directly assayed for TPC according to the Folin-Ciocalteau procedure [2O. The reaction was neutralized with 2 mL of 10% sodium carbonate. The absorbance was measured with a Helios Gamma Spectrophotometer  at 725 nm against a blank after 30 min of reaction at room temperature in a dark place. The TPC was expressed as gallic acid equivalent (GAE) in mg/g of dry weight (d.w.). Measurements were carried out twice.The total polyphenols content (TPC) was determined as follows. Aqueous-organic extracts were isolated as reported by Lisiecka et al. . In a turocedure . In brieNext, 10 g of pasta underwent hydration with 200 mL of hot water (~98 °C). The minimal preparation time (MPT) was achieved after the disappearance of the inner white core of the pasta strand when squeezing it between two Plexiglas plates [After, 10 g of pasta were hydrated with 200 mL of hot water. After the corresponding MPT, the hydrated pasta was rinsed with tap water (20 °C) and drained for 5 min. Both hydrating and rinsing water were collected in a glass beaker and then evaporated in an air oven at 110 °C until constant weight was reached. Water absorption capacity (WAC) and cooking loss (CL) were calculated in triplicate using the following equations :(3)WAC  was applied to calculate hardness (N) as the maximum cutting force using a Warner-Bratzler cutting knife (equipment supplied by Zwick), firmness (N) as the highest peak at first compression, and stickiness (mJ) as the energy needed to remove the working plate from the pasta surface after compression using an Ottawa Texture Measuring System cell (OTMS cell) (equipment supplied by Zwick) [Hardness of dry pasta (in five replications) and firmness and stickiness of hydrated pasta (in two replications) were determined using a Zwick-Roell BDO-FB0.5 TH instrument  with the speed of 3.3 mm/s and the working head of 0.5 kN. Subsequently, TestXpertThe expansion ratio (ER) was calculated by dividing the diameter of the pasta strand (average of 10 replications) by the die diameter.L* values measure lightness (0 = black–100 = white), a* values measure redness  and greenness , and b* values measure yellowness  and blueness .The color of dry and hydrated pasta was evaluated using Lovibond CAM-System 500 Imaging Colorimeter  in 10 replications. Results are expressed using CIE-Lab color scale. Herein, Rice-buckwheat pasta (100 g) was hydrated in hot water for the corresponding MPT, drained and served after 5 min on white plates with no suggestive information to a panel of 15 untrained members educated with requirements for pasta products. Panelists evaluated appearance, color, flavor, taste, and stickiness of pasta by applying a five-point scale  as described by Wójtowicz and Mościcki . The samThe microstructure of dry and hydrated rice-buckwheat pasta was characterized using scanning electron microscopy (SEM). The hydrated pasta was lyophilized before characterization. Pasta samples of 5 mm were mounted on carbon discs and sputter-coated with gold in a vacuum sublimatorK-550X . The scanning electron microscope VEGA LMU  operating at the accelerating voltage of 30 kV was used to characterize the cross-section of samples at different magnifications .R2) and its statistical significance was ascertained by a F-test. Models were considered significant at p values <0.05. Statistica software version 10  was applied to perform statistical differences between mean values at p < 0.05 using one-way analysis of variance (ANOVA), followed by the Tukey’s honestly significant difference (HSD) post hoc test.Statistical analysis was performed using JMP software version 7.0 . The experimental data were evaluated by means of multiple regression analysis and one-way analysis of variance (ANOVA). Validation of the model was expressed by the coefficient of determination  (these were found to be between 0.94 and 0.98).The regression model ectively . Here, pp < 0.01) on CL. This increased with the increase of dough moisture content  on CL. This parameter decreased as barrel temperature increased; in this case, the effect of screw speed was less important .Barrel temperature had a great negative significant effect (mportant b. Giménep < 0.01) by barrel temperature and screw speed. The positive estimate coefficients of barrel temperature and screw speed reveal that hardness of dry pasta increased with increasing barrel temperature .It seems clear from the estimate regression coefficients presented in perature a and scrperature b. Wójtowp < 0.05), while barrel temperature and screw speed had a positive significant effect . Overall, firmness of rice-buckwheat pasta increased when dough moisture content decreased .In contrast, firmness of hydrated pasta increased when barrel temperature and screw speed increased b. Wang ep < 0.05) and negatively by the barrel temperature (p < 0.001). Stickiness of hydrated pasta decreased as barrel temperature increased , ash (1.58%), fiber (4.57%), and TPC (1.85 mg GAE/g) contents than rice flour  . BouaslaIn our experiment, incorporation of buckwheat flour led to an increase of TPC in rice-buckwheat pasta (0.40 mg GAE/g) 10 times more than that of rice flour (0.04 mg GAE/g). Phenolic acids  and flavonoids  make up most of the polyphenol classes found in buckwheat . The extIn addition, very low-fat content (0.16%) has been noticed for rice-buckwheat pasta, as compared to the separate raw materials . Similar observations have been reported for wheat-based and rice-based pasta manufactured by extrusion-cooking ,21. ThisHot water hydration for 8.5 min was sufficient for rice-buckwheat pasta to have the proper consistency. This differs from conventional cooking. The comparison of our results with previous studies using the same method of hydration in hot water revealed that rice-buckwheat pasta had a MPT slightly higher than the time reported by Bouasla et al.  for riceWater absorption capacity of rice-buckwheat pasta (237.77 g/100g) was also significantly higher than that reported for rice pasta (181.53 g/100 g). This effect could be explained by the higher water-binding capacity of buckwheat starch , as wellRice-buckwheat pasta had significantly higher CL (5.23%) than rice pasta (3.57%). This increase in CL could be related to the presence of fiber, as this weakens the starch network in both gluten-free matrices and gluten-based matrices . HoweverOur work showed that the expansion ratio of rice-buckwheat pasta 1.21) was significantly lower than that of rice pasta (1.55). This may be due to the low amount of total starch and high amount of protein and fiber in buckwheat flour, which limits the expansion of pasta products  was sign. Yet, asPasta color is considered an essential parameter for evaluating pasta quality . Both drDry rice-buckwheat pasta showed significant lower hardness (4.05 N) than rice pasta (12.12 N). This effect could be related to the lower diameter of rice-buckwheat pasta, compared to rice pasta, and to the presence of fiber fractions from buckwheat flour. These fiber fractions could weaken pasta structure by the formation of discontinuities or cracks inside the pasta strand .Adding buckwheat flour into the pasta recipe increased the firmness of the hydrated pasta. Firmness values differed significantly, with values of 199.5 N for rice pasta and 271.0 N for rice-buckwheat pasta. This can be explained by the higher protein content in rice-buckwheat pasta, compared to rice pasta and the formation of more firm structure after hot water hydration.The incorporation of buckwheat flour increased significantly the stickiness of hydrated pasta . This increase in pasta stickiness could be the result of higher fiber content and/or higher amount of compounds leached from the surface of rice-buckwheat pasta during hot water hydration, as confirmed by higher cooking loss compared to rice pasta. Alamprese et al.  showed tFor all of the sensory attributes of hydrated pasta, rice-buckwheat products received higher scores than rice pasta . When apThe replacement of 50% of starchy components by pseudo-cereals such as buckwheat in bread  or 25% oThe microstructure of dry and hydrated optimum rice-buckwheat pasta was observed with a scanning electron microscopy. Herein, dry rice-buckwheat pasta demonstrated a smooth surface a,b. AfteA homogenous and compact inside structure was noted during the observation of the cross-sectional microstructure of dry pasta a–c. ThisThe effect of extrusion-cooking parameters on the quality of rice-buckwheat gluten-free pasta was studied using a Box-Behnken design. The results indicated that moisture content, barrel temperature, and screw speed had different effects on the cooking and textural properties of gluten-free pasta. Barrel temperature significantly affected all tested properties. An increase in barrel temperature induced a decrease in cooking loss and stickiness and an increase in hardness and firmness, while the reverse effect was observed for moisture content except for hardness. Screw speed also showed a significant positive effect, albeit only on hardness and firmness.In our work, an optimum gluten-free pasta was produced at 30% of moisture content, 120 °C for temperature, and 80 rpm for screw speed and was then evaluated for its characteristics. This optimum rice-buckwheat pasta showed good quality, specifically for high nutritional composition, low cooking loss and stickiness, and acceptable score for all sensory attributes and overall acceptability, with homogeneous and compact microstructure. Based on the obtained results, gluten-free rice-buckwheat pasta would constitute as a good alternative for persons afflicted with celiac disease.


Enzymatic biosensors are widely used in clinical diagnostics, and electrode materials are essential for both the efficient immobilization of enzyme and the fast electron transfer between the active sites of enzyme and electrode surface. Electrode materials with a hierarchically porous structure can not only increase the specific surface area but also promote the electron transfer, facilitating the catalysis reaction. Block copolymer is a good candidate for preparation of film with a hierarchically porous structure due to its unique characteristics of self-assembly and phase separation. In the current work, hierarchically porous block copolymer film containing both micropores and nanopores was prepared by spinodal decomposition induced phase separation. The resultant copolymer film was adopted as the electrode material to immobilize glucose oxidase (GOx) for construction of an enzyme biosensor. Scanning electron microscopy (SEM), contact angle (CA) measurements, and Fourier-transform infrared (FTIR) and electrochemical impendence spectroscopy (EIS) were adopted to investigate the microstructure of the as-developed biosensor. Results demonstrated that the hierarchically porous block copolymer film offered a favorable and biocompatible microenvironment for proteins. These as-prepared glucose biosensors possessed a wide linear range (10–4500 μM), a low detection limit (0.05 μM), quick response (2 s), excellent stability, and selectivity. This work demonstrates that hierarchically porous block copolymer film is a good matrix candidate for the immobilization of the enzyme and provides a potential electrode material to construct novel biosensors with excellent performance. A biosensor, which consists of confident biological component coupled with a transducer device, can convert a biochemical signal into an amplified electrical signal ,2,3. Cur−1·cm−2, a low detection limit of 20 nM (S/N = 3), excellent selectivity, reproducibility, and stability. On the other hand, porous structure, especially the hierarchically porous structure containing micropores and nanopores, is introduced into the electrode material in order to increase the reaction sites and enhance the electron transfer [In general, it is crucial to efficiently immobilize enzymes while at the same time maintaining effective electron transfer between the active sites of enzymes and the electrode surface. Various conducting materials have been used as electrode materials, like graphene , carbon transfer .block-poly(2-hydroxyethyl methacrylate) (PS-b-PHEMA) film possessing a perfect hexagonal lattice structure, with apore diameter of 24.7 ± 2.5 nm. Moreover, vertically oriented pores were present on the top surface of the asymmetric film.Block copolymers provide many attractive possibilities in designing hierarchically porous materials ,17,18. SIn the present work, we prepared hierarchically porous block copolymer film containing micropores and nanopores by macro- and meso-phase separation, which was induced by spinodal decomposition. Subsequently, the resultant film was adopted as the electrode material to immobilize glucose oxidase (GOx). The as-developed glucose biosensor displayed a long linear range (10–4500 μM), a low detection limit (0.05 μM), and quick response (2 s). Moreover, the selectivity and stability were also investigated. This work illustrates that the hierarchically porous block copolymer films as electrode materials have great potential applications in the field of high performance enzyme biosensors.block-poly(4-vinyl pyridine) (PS9w-b-P4VP5w) was obtained from Polymer Source Inc. ; polyethylene glycol  was provided from Aladdin Chemical Reagent Co.; anisole was received by Lingfeng Chemical Reagent Co. ; glucose oxidase (GOx) from Aspergillus niger  was supplied by Sigma-Aldrich Chemical Co., Ltd. ; d-glucose, KH2PO4, and K2HPO4 were purchased from Sinopharm Chemical Reagent Co. . d-glucose solution was prepared one day in advance and mutarotated at room temperature. All liquid reagents used for the preparation of block copolymer film were carefully dried over molecular sieves (3A). All of the aqueous solutions in the process of electrochemical measurements were prepared with twice-distilled water. All the other chemical reagents were analytical grade and used without further purification. The platinum (Pt) electrodes were successively polished to a mirror-like surface with 0.5 μm and 50 nm alumina slurry and then rinsed thoroughly by twice-distilled water. To get rid of the loosely adsorbed alumina slurry, the electrodes were washed with anhydrous ethanol for 3 min in an ultrasonic bath, rinsing thoroughly by twice-distilled water followed by drying with a stream of nitrogen. Phosphate buffer (PB) solution was transferred to 6.5 with 0.1 M of KH2PO4 and K2HPO4 in total. Polystyrene-b-P4VP film and GOx/PS-b-P4VP composite film were observed with scanning electron microscopy . Prior to SEM observation, the films were sputter coated for 35 s with gold to avert charge accumulations during the test. The measurements for the contact angles (CA) were carried out with a video optical contact angle measuring instrument (OCA20). Attenuated total reflectance Fourier transform infrared  spectrum was recorded in the range of 500−3600 cm−1 on a Cary610 ATR-FTIR spectrometer. Electrochemical impedance spectroscopy  was adopted to determine the charge transfer resistance of bare Pt electrode, PS-b-P4VP/Pt, and GOx/PS-b-P4VP/Pt. EIS was studied in 0.1 M PB containing 5 mM Fe(CN)63+/4+ and 0.1 M KCl with a frequency range of 0.05 Hz to 100 kHz. Electrochemical workstation (CHI660A) was used for amperometric measurements. A three-electrode system with a working electrode , a counter electrode , and a reference electrode  was employed for the electrochemical measurements.The morphologies of PS-b-P4VP and 0.28 g of PEG were dissolved in 5.4 g of anisole at 40 °C for 12 h to form a homogeneous solution, followed by filtration with a 0.22 µm PTFE syringe filter to remove impurities. Then, the polymer solution was dropped onto the surface of electrode and subsequently heated at 130 °C for 2.5 h. The polymer solution was solidified with evaporation of the solvent, and a white copolymer film was generated on the electrode surface. Then the copolymer film deposited on the electrodes was immersed in ethanol and gently stirred for 2 h to remove the PEG from the BCP/PEG co-assembled structures and the macro-phase separated PEG-rich domain. Finally, the hierarchically porous PS-b-P4VP film was obtained. The schematic demonstration for the fabrication of hierarchically porous block copolymer film is shown in 0.32 g of PS-b-P4VP/Pt was rinsed thoroughly with twice-distilled water to remove the loosely adsorbed GOx molecules. All the as-prepared electrodes were stored at 4 °C in a refrigerator before use. All the experiments were carried out at room temperature, unless otherwise specified.1.5 mg GOx was dissolved with 1.5 mL PB solution (pH 6.5) to form a uniform solution. Afterwards, 8 μLof the solution was dropped on the surface of the pretreated Pt electrode; and dried in a refrigerator. Then the electrodes were exposed to 25% glutaraldehyde vapor for 15–20 min so as to avoid the leakage of the immobilized enzyme molecules. Moreover, the GOx/PS-b-P4VP film before and after immobilization of GOx are shown from the SEM images in b-P4VP film  and amide II (1600−1500cm−1) bands of protein [−1 in curve b are associated with the characteristic absorption peaks of alkyl I (2925 cm−1) and alkyl II (2850 cm−1) for the block copolymer film. The absorption peaks at 1599, 1494, and1451 cm−1 are ascribed to the stretching vibration peaks of benzene ring. The absorption peaks at 752 and 694 cm−1 are assigned to pyridine and benzene, respectively. Compared with curve a and b, the GOx/PS-b-P4VP composite film possesses the characteristic absorption peaks of both GOx and PS-b-P4VP (curve c) as mentioned above, indicating that GOx has been successfully immobilized on the PS-b-P4VP film. In addition, the peaks for amide I and II bands shifts from 1639 to 1658 cm−1, indicating the intermolecular interaction between enzyme and the hierarchically porous PS-b-P4VP film.The ATR-FTIR spectroscopy was utilized to characterize the interaction of the immobilized GOx and the matrix, which is shown in  protein . The absRs comprises the resistance of the electrode and electrolyte resistance, dIC is the constant phase element related to the double layer capacitance, and the ctR represents the charge transfer resistance. All electrodes after modification display a semicircle resulting from ctR in the high frequency region, followed by a linear region known as Warburg impedance wZ in the low-frequency region, and the wZ corresponds to the diffusion process. The value of ctR can be obtained via the diameter of semicircle. The ctR values of bare Pt, PS-b-P4VP/Pt, andGOx/PS-b-P4VP/Pt are 73, 252, and 2453 Ω, respectively. Compared with the bare Pt, PS-b-P4VP/Pt has a relatively slight increase in the ctR, indicating that the PS-b-P4VP film retains the great transference of electron, which may be ascribed to the hierarchically porous structure. In addition, an obvious increase in the resistance and the weak diffusion process of the GOx/PS-b-P4VP/Pt can be observed when GOx is immobilized in the copolymer film, which results from the hindrance of the macromolecular structure of GOx to the electron transfer. This result is in agreement with that of Shan [b-P4VP film.Electrochemical impedance spectroscopy (EIS) is a powerful electrochemical method that can provide electrochemical information of the electrodes. It can reflect the electron transfer resistance and the diffusion of redox-active compounds within solution and electrode interface . Figure  of Shan  and Su [ of Shan . Thence,The performance of enzyme biosensor relates to many factors, for example, the thickness of bio-composite film and external factors. In this work, the thickness of bio-composite film mainly relies on the polymer solution and the amount of enzyme. In general, a higher concentration of polymer results in a thicker film that may hinder the fastelectron transfer. −2 with the increase of temperature from 20 to 40 °C. Then the current response descends when the temperature rises to 60 °C. We carried out the following experiments at 25 °C, because the protein unfolding occurred and long term stability of the biosensor was sacrificed at a relatively high temperature. However, the measurement of relationship between current response and temperature is not meaningless. The apparent activation energy (Ea) of the reaction can be calculated according to the Arrhenius relationshipExternal factors—such as pH, temperature and applied potential—can also influence the electrochemical performance of enzyme biosensors. Among them, pH and temperature determine the enzymatic electrocatalytic activity and stability, while the applied potential affects the sensitivity and selectivity of the biosensor. The influence of pH on current response is displayed in R is the molar gas constant, T is the thermodynamic temperature and A is pre-exponential factor. lnk could be replaced with lnI. Then aE was obtained by the slope of the line  was calculated to be 3 mM for the glucose biosensor via Lineweaver–Burk plot. For the reason that Km demonstrates the activity of enzyme and affinity between enzyme and immobilization matrix, such low value of Km indicates that the hierarchically porous PS-b-P4VP film provides a biocompatible micro-environment for enzyme; and these materials are considered as a potential electrode material to immobilize enzyme.As is known, GOx can catalyze the oxidation of glucose to produce gluconolactone and hydrogen peroxide ,25. The inset in A. The quThe selectivity of the biosensor for quantitative detection of glucose was examined by using glucose, fructose, lactose, and NaCl as analytes. As shown in b-P4VP film retained its native structure and bioactivity, and hence exhibited fast amperometric response to glucose. Compared with previously reported glucose biosensor, the as-developed biosensor exhibited more surprising performances in response time (2 s), linear range (10–4500 μM), and detection limit (0.05 μM). This work illustrates that the hierarchically porous block copolymer film is a great support matrix for immobilization of enzyme and provides a potential electrode material to construct novel biosensors with excellent performance. In this work, block copolymer film with a hierarchically porous structure was prepared for the immobilization of enzyme. The results proved that the hierarchically porous structure offered a bio-compatible microenvironment for the immobilization of enzyme and enhanced the direct electron transfer between the enzyme and electrode surface. Then, a glucose biosensor was constructed based on the porous matrix as the electrode material. The GOx in PS-